In the field of fluorescence fluctuation microscopy (FFM), fluorescence or phosphorescence from a sample can be used to study properties of organic or inorganic substances. For example, the substance to be tested contains fluorescence-tagged molecules, and it is illuminated with laser light of a wavelength chosen to excite the fluorescence. The fluorescence generated by the sample is detected, by means of an optics system comprising a suitable detector. Measurements can then be made of the sample in order to permit the quantification of molecular dynamics and molecular concentration within the sample. For example, quantities such as the mean number of molecules <N> within the observation volume, and/or the diffusion time τD of the molecules, can be estimated.
Fluorescence correlation spectroscopy (FCS) is a particular example of FFM, in which correlation analysis is applied to fluctuations in the fluorescence intensity.
A problem with existing FFM techniques is that, because measured quantities are very dependent on the optically defined observation volume, there tends to be a relatively large variation in the measurements of the same substance obtained during different acquisition series, or in different environments.